Promoter Amplification, Sequence Comparison of PPARs in Duck (Anas platyrhynchos), and Cluster Analysis with Transcription Factors in Expression Profiles
Abstract:Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily, and there are 3 subtypes including α, β/δ and γ, and all PPARs members play a crucial role in cell growth and differentiation. In order to investigate their distinct functions in the process of skeletal muscle development, PPARs promoter sequences in duck (Anas platyrhynchos) were cloned using PCR and were analyzed using several bioinformatics tools. The mRNA expression profiles of PPARs and their common transcription factors in the breast and leg muscle of duck during embryonic stage were detected using qRT-PCR. The PCR results showed that PPARα, PPARβ and PPARγ promoter sequences of duck were 2 526, 1 631 and 2 942 bp,respectively, and GenBank accession were KX845431, KX845432 and KX845433, respectively. Their homology reached 99%, 97% and 97% with the predicted sequences of duck genome in NCBI, respectively. All PPARs promoters in duck shared higher homology compared with chicken (Gallus gallus) near the translation initiation sites through analyzing the cloned promoter sequences. The typical cis acting elements (CAAT-box and TATA-box) had been predicted to exist in the promoter region of PPARs, and common binding sites of transcription factors (specificity protein 1 (Sp1), nuclear factor kappa B (NF-κB) and CCAAT/enhancer binding protein alpha (C/EBP-α)) were predicted to exist in all duck PPARs members. Moreover, PPARα and PPARγ shared 9 common transcription factors, and PPARβ shared 8 with PPARγ, while PPARα and PPARβ shared the least number of common transcription factor. The qRT-PCR results showed that PPARα, PPARβ and PPARγ expressed in breast and leg muscle tissues of duck. Clustering results showed that the expression patterns of PPARβ and PPARγ were more similar compared with PPARα in leg muscle. It could be seen that the functions of the 2 genes were more similar in the process of duck skeletal muscle development. Besides, in breast muscle, the expression pattern of transcription factor NF-κB and PPARβ gene was consistent. PPARs members all participated in the regulation of skeletal muscle development, and the function of PPARβ might be larger; the function of PPARβ and PPARγ in skeletal muscle development might be similar. In addition, the NF-κB might control the expression of PPARβ in skeletal muscle. The results provide theoretical basis for the expression and function identification of PPARs gene.
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